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| United States Patent |
6,083,719
|
|
Momparler
, et al.
|
July 4, 2000
|
Cytidine deaminase cDNA as a positive selectable marker for gene
transfer, gene therapy and protein synthesis
Abstract
The present invention relates to a DNA sequence for the human cytidine
deaminase that has been engineered into an eukaryotic expression vector,
thereby permitting cytidine deaminase expression in mammalian cells.
Cytidine deaminase expression confers resistance to cytosine nucleoside
analogs, such as cytosine arabinoside, and can be used as a positive
selectable marker. The expression of cytidine deaminase in cells protects
them from the toxic effects of cytosine nucleoside analogs. Such a
resistance provides applications for gene therapy of malignant, immune and
viral diseases. A bacterial expression vector containing the gene can be
used to produce cytidine deaminase in large quantities.
| Inventors:
|
Momparler; Richard L. (Montreal, CA);
Laliberte; Josee (Chapel Hill, NC);
Cournoyer; Denis (Montreal, CA);
Eliopoulos; Nicoletta (Montreal, CA)
|
| Assignee:
|
Hopital Sainte-Justine (Montreal, CA)
|
| Appl. No.:
|
968768 |
| Filed:
|
October 27, 1997 |
| Current U.S. Class: |
435/69.1; 435/183; 435/227; 435/320.1; 435/325; 435/375; 536/23.1; 536/23.5 |
| Intern'l Class: |
C12N 005/10; C12N 015/09; C12N 015/79; C12N 015/63 |
| Field of Search: |
435/69.1,320.1,375,7.2,325,354,363,366,183,227
536/23.1,23.5
|
References Cited
U.S. Patent Documents
| 5399346 | Mar., 1995 | Anderson et al. | 424/93.
|
| Foreign Patent Documents |
| WO 92/07943 | May., 1992 | WO.
| |
Other References
Laliberte et al., 1994, Cancer Res. 54: 5401-5407.
Markowitz et al., 1988, J. Virol. 62: 1120-1124.
Corey et al., 1990, Blood 75: 337-343.
Podda et al., 1992, Proc. Natl. Acad. Sci. USA 89: 9676-9680.
Ward et al., 1994, Blood 84: 1408-1414.
Chabner et al., 1990, In Cancer Chemotherapy, Principles and Practice,
Lippincott Company, pp. 154-179.
Cepko 1989, In Current Protocols in Molecular Biology, pp. 9.10.1-9.11.12,
Ausubel et al., Eds., Wiley & Sons.
Dwarki et al., 1995, Proc. Natl. Acad. Sci. USA 92: 1023-1027.
Chabot et al., 1983, Biochem. Pharmacol. 32: 1327-1328.
Bouffard et al., 1993, Biochem. Pharmacol. 45: 1857-1861.
Ghattas et al., 1991, Mol. Cell. Biol. 11: 5848-5859.
Boyum et al., 1994, Exp. Hematology 22: 208-214.
Kuhn et al., 1993, Biochem., Biophys. Res. Com. 190: 1-7.
Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd, Cold
Spring Harbor Laboratory Press, N.Y.
Steuart, et al. "Cytidine deaminase and the development of resistance to
arabinosyl cytosine," Nature, vol. 233 (38): 109-110, Sep. 22, 1971.
Ohe, et al. "Combination effect of vaccination with IL-2 and IL-4 cDNA
transfected cells on the induction of a therapeutic immune response
against Lewis Lung carcinoma," Int. J. Cancer, vol. 53: 432-437, 1993.
Miller, A.D. "Progress toward human gene therapy," Blood, vol. 76(2):
271-278, Jul. 15, 1990.
Coghlan, A. "Gene dream fades away," New Scientist, pp. 14-15, Nov. 25,
1995.
Brown, D. "Gene Therapy `Oversole` by Researchers, Journalists," The
Washington Post, A22, Dec. 8, 1995.
Roemer, et al. "Concepts and Strategies for human gene therapy," Eur. J.
Biochem., vol. 208: 211-225, 1992.
|
Primary Examiner: Hauda; Karen M.
Attorney, Agent or Firm: Swabey Ogilvy Renault, Cote; France
Parent Case Text
This application is a continuation of application Ser. No. 08/509,138 filed
on Jul. 31, 1995 now abandoned.
Claims
We claim:
1. A eukaryotic expression vector comprising the human cytidine deaminase
nucleic acid sequence set forth in SEQ ID NO: 1, wherein said human
cytidine deaminase nucleic acid sequence is operably linked to
transcription regulatory sequences to confer expression of said human
cytidine deaminase by a eukaryotic host cell upon transfection with said
eukaryotic expression vector.
2. The eukaryotic expression vector of claim 1, wherein the eukaryotic host
cell is a mammalian cell which is resistant to cytosine arabinoside or
related cytosine analogs upon expression of said human cytidine deaminase.
3. The eukaryotic expression vector of claim 2, comprising in addition to
cytidine deaminase at least one additional nucleic acid sequence
expressible in said mammalian cell.
4. The eukaryotic expression vector of claim 3, wherein the additional
nucleic acid sequence encodes a dominant selectable marker selected from
the group consisting of a dehydrofolate reductase gene, a multidrug
resistance gene, a gpt gene and a neomycin gene.
5. The eukaiyotic expression vector of claim 1, which is retroviral
expression vector pMFG-CD.
6. The eukaryotic expression vector of claim 1, comprising in addition to
cytidine deaminase, at least one additional nucleic acid sequence
expressible in said host cell.
7. The eukaryotic expression vector of claim 6, which is retroviral
expression vector pMFG-CD.
8. The eukaryotic expression vector of claim 3, which is retroviral
expression vector pMFG-CD.
9. An in vitro method of conferring resistance on a mammalian cell to
cytosine arabinoside or related cytosine analogs comprising the steps of:
a) transfecting into a producer cell the expression vector of claim 2,
thereby producing retroviral particles;
b) transducing a population of mammalian cells with the retroviral vector
particles produced by step (a); and
c) selecting cytidine deaminase-expressing cells from said transduced cells
of step (b) thereby obtaining mammalian cells which express human cytidine
deaminase and are resistant to cytosine arabinoside or related cytosine
analogs.
10. A eukaryotic cell which comprises the eukaryotic expression vector of
claim 1.
11. The eukaryotic cell of claim 10, which is a mammalian cell.
12. An isolated nucleic acid molecule comprising the nucleic acid sequence
set forth in SEQ ID NO: 1.
Description
BACKGROUND OF THE INVENTION
i) Field of the Invention
This invention relates to a human nucleic acid for cytidine deaminase that
has been engineered into eukaryotic and bacterial expression vectors, to
the expression of human cytidine deaminase by mammalian cells and
bacterial cells, and to cells expressing human cytidine deaminase. The
invention further relates to methods and gene therapies that employ the
dominant selectable marker, cytidine deaminase, which has the ability to
inactivate a toxic antimetabolite such as cytosine arabinoside by
deamination to uracil arabinoside.
ii) Description of the Prior Art
Selectable markers are important tools in the study, regulation and
function of genes and are potentially important in gene transfer
therapies. Conferring a unique resistance to a cytotoxic agent enables the
skilled artisan the ability to select genetically altered cells from a
mixed population.
The enzyme cytidine deaminase (CD) has the ability to catalyze the
deamination of cytosine arabinoside, an antimetabolite that is toxic to
mammalian cells, to uracil arabinoside which is non-toxic at
pharmacological concentrations (Chabner, et al., 1990, In Cancer
Chemotherapy: Principles and Practice pp. 154-179, Lippincott Company). CD
can also inactivate, by deamination, other cytosine nucleoside analogs
that are currently used as anticancer agents (Chabot et al., 1983,
Biochem. Pharmacol. 32: 1327-1328; Bouffard et al., 1993, Biochem.
Pharmacol., 45: 1857-1861). In mammalian cells cytosine arabinoside
(ARA-C), is metabolized to ARA-CMP, ARA-CDP and ARA-CTP, the latter
nucleotide analog is incorporated into DNA, producing a potent inhibition
of DNA synthesis and resulting in growth inhibition and cell death
(Chabner, et al., 1990, In Cancer Chemotherapy: Principles and Practice pp
154-179, Lippincott Company).
The availability of a dominant selectable marker to cytosine analogs, would
be a significant advantage for the skilled artisan in the field of
molecular biology, cell biology and gene transfer technology in
eukaryotes.
The efficacy of treatments of patients with cytosine nucleoside analogs,
suffers from the relatively high doses of cytosine analogs required and
the accompanying side effects resulting from normal cell cytotoxicity.
Indeed, bone marrow suppression is the major dose-limiting toxicity
produced by intensive chemotherapy with ARA-C and related cytosine
nucleoside analogs.
It would thus be advantageous for the patient and clinician if susceptible
cells, such as bone marrow cells could be protected from the toxic effects
of the cytosine analog. This protection could permit an increase in the
therapeutic index of the drug. In addition, it could permit an increase in
the dose administered without substantially increasing the side effects of
the drug. A similar approach has been reported for the establishment of
methotrexate-resistant bone marrow cells (Corey, et al., 1990, Blood 75:
337-343).
A partial cDNA for human CD has been isolated and the expression of
cytidine deaminase demonstrated in bacteria but not in eukaryotic cells
(Kuhn, et al., 1993, Biochem. Biophys. Res. Com. 190: 1-7). Recently, the
full-length human cDNA has been isolated and its DNA and amino acid
sequence determined (Laliberte et al., 1994, Cancer Res. 54: 5401-5407).
Having the full-length human cDNA for CD, it would be advantageous to
engineer vectors permitting expression of cytidine deaminase in cells. It
would also be beneficial to obtain bone marrow cells expressing human CD
and implant them in a patient suffering form an immune disease, prior to
the treatment of this patient with cytosine analogs. This kind of gene
therapy and others, could be beneficial for the treatment of diseases
including but not limited to acute T-cell disorders, rheumatoid arthritis,
and autoimmune diseases. Gene therapy using CD could also be beneficial
for preventing graft rejection.
Cytidine deaminase has been reported to inhibit the proliferation of
myeloid hematopoietic cells (Boyum et al., 1994, Exp. Hematology, 22:
208-214). This enzyme could thus have therapeutic use in the treatment of
certain types of leukemia.
It would be advantageous to obtain large amounts of human cytidine
deaminase to test the therapeutic potential of the enzyme. In addition, it
would be beneficial to obtain cells expressing CD, as they could be used
to test for inhibitors or up-regulators of cytidine deaminase activity.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a new positive selectable
marker, resistance to cytosine analogs, through the expression of a
nucleic acid for human CD. When integrated into a eukaryotic expression
vector and expressed in mammalian cells, cytidine deaminase confers to the
transfected cell a unique resistance to the cytotoxic effects of ARA-C and
related cytosine nucleoside analogs.
It is a further object of the present invention to provide a host cell
comprising a nucleic acid construct which permits expression of human
cytidine deaminase. The invention also seeks to provide expression vectors
harboring the cytidine deaminase nucleic acid segment of the invention in
an expressible from, and cells transformed with same. Such cells can serve
a variety of purposes such as in vitro models for the function of CD as
well as for screening pharmaceutical compounds that could regulate the
expression of the CD gene or the activity of the protein encoded
therefrom. An expression vector harboring the CD nucleic acid segment or
part thereof, can be used to obtain substantially pure protein or a
peptide fragment therefrom. The purified enzyme or peptide fragment may be
used as a therapeutic agent to inhibit proliferation of certain types of
leukemia. An example of a construct expressing CD is the DNA plasmid
construct designated pMFG-CD.
Well-known vectors can be used to obtain large amounts of the protein which
can then be purified by standard biochemical methods based on charge,
molecular weight, solubility or affinity of the protein or alternatively,
the protein can be purified by using gene fusion techniques such as GST
fusion, which permits the purification of the protein of interest on a
gluthathion column. Other types of purification methods or fusion proteins
could also be used.
It is also contemplated to provide shuttle vectors, comprising a nucleic
acid sequence encoding human cytidine deaminase capable of expression in
eukaryotic cells as well as sequences expressible in microorganisms. Such
vectors are designed to confer a selective advantage in both the
eukaryotic and prokaryotic cells harboring them.
Antibodies both polyclonal and monoclonal can be prepared from the protein
encoded by the CD nucleic acid segment of the invention. Such antibodies
can be used for a variety of purposes including affinity purification of
the CD protein and to determine the level of expression of the cytidine
deaminase protein in cells. The latter could be of use for the indirect
determination of the resistance of cancer cells to ARA-C and related
cytosine analogs for example. It is also contemplated that sequences from
the human CD nucleic acid sequence, variants or homologs thereof, or
oligonucleotides derived from these nucleic acid sequences, could also be
used to determine the level of expression of cytidine deaminase in cells.
Such an information could, for example, be used to orient the
chemotherapeutic treatment profile of a patient.
It is yet a further object of this invention to provide in vitro methods
for the selection of clones in a mixed population of cells. These methods
can be used for gene transfer. In addition, selected cell clones can be
used in vivo for gene transfer therapies.
It is still a further object of this invention to provide therapeutic
methods for malignant, immune and viral diseases, based on an expression
of a human nucleic acid encoding CD or a variant thereof retaining
cytidine deaminase activity, and a chemotherapeutic treatment with
cytosine nucleoside analogs.
More specifically, it is another object of the present invention to provide
a cytidine deaminase positive selection system for gene transfer therapies
comprising the steps of inserting the nucleic acid sequence comprising the
human cytidine deaminase gene or variant thereof in a eukaryotic
expression vector, along with an exogenous nucleic acid sequence to be
expressed, transfecting this construct into a host genome and treating the
host or parts thereof with ARA-C or related cytosine nucleoside analogs in
pharmacologically acceptable doses, so as to select cells that have
integrated the construct into their genome. Examples of products encoded
by exogenous nucleic acid sequences include but are not limited to tumour
suppressor genes, growth factors and single chain antibodies to specific
proteins.
In a further embodiment, it is an object of the invention to provide a
method of administering allogenic or autologous bone marrow cells into a
patient comprising the steps of treating the bone marrow cells with a
cytidine deaminase construct packaged into a vector in such a manner that
the construct will preferentially confer resistance to ARA-C and related
cytosine nucleoside analogs to the bone marrow cells. Administering the
treated bone marrow cells to a patient and subsequently administering
toxic doses of ARA-C or related cytosine nucleoside analogs such that
tumor cells or abnormal lymphocytes are destroyed, whereas the
transplanted bone marrow cells survive, thereby reducing the bone marrow
toxicity produced by these analogs.
In addition, it is an object of the present invention to provide the human
cytidine deaminase nucleic acid, sequences hybridizing thereto under high
stringency conditions, a corresponding sequence within the scope of the
degeneracy of the genetic code or a functional variant of such a nucleic
acid sequence.
In addition, it is an object of this invention to provide retroviral
particles, containing the human cytidine deaminase nucleic acid. These
retroviral particles can be used to infect cells, such as normal
hematopoietic stem cells in vitro, to confer resistance to ARA-C and
related cytosine nucleoside analogs. Through infusion of these cells into
a patient, the patient now possesses cells that are protected from the
cytotoxic effects of a chemotherapeutic treatment with cytosine analogs
such as ARA-C.
The designation functional variant is to be interpreted as meaning that the
variant retains the biological activity of the protein from which it might
originate. As used herein, the term "oligonucleotide" includes both
oligomers of ribonucleotides and oligomers of deoxyribonucleotides.
The designation related cytosine analogues as used herein is meant to
include without being limited thereto: 2,2'-difluorodeoxycytidine and
5-aza-2'-deoxycytidine.
The term high stringency hybridization conditions, as used herein and well
known in the art, includes, for example: 5.times.SSPE (1.times.SSPE is 10
mM Na-phosphate, pH 7.0; 0.18 M NaCl; 1 mM Na.sub.2 EDTA),
5.times.Denhardt's solution (from a 100.times.solution containing 2% BSA,
2% Ficoll, 2% polyvinyl pyrollidone), 0.1% SDS, and 0,5 mg/ml denatured
salmon sperm DNA, at 65.degree. C. Other conditions considered stringent
include the use of formamide. An example of washing conditions for the
blot includes, as a final stringency wash, an incubation of the blot at
65.degree. C. in 0.1.times.SSPE, 0.1% SDS for 1 hour.
From the specification and appended claims, it should be understood that
the term nucleic acid should be taken in a broad sense so as to include,
while not being limited thereto, the cDNA and the gene encoding CD. In
addition, the term gene as used herein should be interpreted so as to
include the cDNA thereof. The term pharmaceutical composition should be
interpreted as including veterinary compositions.
In accordance with one aspect of the invention there is provided a
eukaryotic expression vector comprising the human cytidine deaminase
nucleic acid sequence as set forth in SEQ. ID NO: 1, a complement thereof,
a functional variant thereof due to the degeneracy of the genetic code or
a nucleotide sequence which hybridizes to SEQ. ID NO: 1, complement
thereof, or variant thereof under stringent conditions, wherein the
cytidine deaminase nucleic acid sequence is functionally positioned in the
eukaryotic expression vector so as to be expressed in a eukaryotic host
cell.
In accordance with another aspect of the invention there is provided a
method of conferring resistance to cytosine arabinoside or related
cytosine analogs to a eukaryotic cell, the method comprising the steps of:
a) introducing into the eukaryotic cell, a DNA construct comprising the
human cytidine deaminase nucleic acid sequence as set forth in SEQ. ID NO:
1, a complement thereof, a functional variant thereof due to the
degeneracy of the genetic code or a nucleotide sequence which hybridizes
to SEQ. ID NO: 1, complement thereof, or variant thereof under stringent
conditions, wherein the cytidine deaminase nucleic acid sequence confers
resistance to cytosine arabinoside or related cytosine analogs when
expressed in the eukaryotic cell; b) growing the eukaryotic cell of a)
under conditions conducive to expression of said human cytidine deaminase
nucleic sequence in the presence of cytosine arabinoside or related
cytosine analogs at a concentration which is toxic to the eukaryotic cell
not expressing the nucleic acid sequence; and c) selecting cytidine
deaminase-expressing cells which are resistant to cytosine arabinoside or
related cytosine analogs.
In accordance with still another aspect of the invention there is provided
a eukaryotic cell harboring the eukaryotic expression vector comprising
the human cytidine deaminase nucleic acid sequence as set forth in SEQ. ID
NO: 1, a complement thereof, a functional variant thereof due to the
degeneracy of the genetic code or a nucleotide sequence which hybridizes
to SEQ. ID NO: 1, complement thereof, or variant thereof under stringent
conditions, wherein the cytidine deaminase nucleic acid sequence is
functionally positioned in the eukaryotic expression vector so as to be
expressed in a eukaryotic host cell.
In accordance with yet another aspect of the invention there is provided a
DNA construct comprising the human cytidine deaminase nucleic acid
sequence as set forth in SEQ. ID NO: 1, a complement thereof, or a
functional variant thereof due to the degeneracy of the genetic code, as
well, there is provided a bacterial cell harboring such a DNA construct.
In accordance with a still further aspect of the invention there is
provided a method of animal gene therapy which comprises: a) introducing
into a eukaryotic cell sensitive to cytosine arabinoside or related
cytosine analogs, a DNA construct comprising the human cytidine deaminase
nucleic acid sequence as set forth in SEQ. ID NO: 1, a complement thereof,
a functional variant thereof due to the degeneracy of the genetic code or
a nucleotide sequence which hybridizes to SEQ. ID NO: 1, complement
thereof, or variant thereof under stringent conditions, wherein the
cytidine deaminase nucleic acid sequence confers resistance to cytosine
arabinoside or related cytosine analogs when expressed in the eukaryotic
cell; b) introducing the cells of a) into the animal; and c) treating the
animal locally or systematically with a pharmacologically acceptable dose
of cytosine arabinoside or related cytosine analogs; such that the
eukaryotic cells having been rendered resistant to a treatment with
cytosine arabinosides or related cytosine analogs are substantially
protected from the cytotoxic effects thereof, and thereby reduce undesired
side effects associated with said treatment.
In accordance with yet another aspect of the invention, there is provided a
eukaryotic expression vector containing a dominant selectable marker
capable of expression in a eukaryotic cell comprising: a) the nucleic acid
sequence as set forth in SEQ. ID NO: 1, a complement thereof, a functional
variant thereof due to the degeneracy of the genetic code or a nucleotide
sequence which hybridizes to SEQ. ID NO: 1, complement thereof, or variant
thereof under stringent conditions, wherein the cytidine deaminase nucleic
acid sequence confers resistance to cytosine arabinoside or related
cytosine analogs when expressed in the eukaryotic cell; b) a pBR322 origin
of replication; and c) a promoter sequence.
In accordance with an additional aspect of the invention there is provided
a dominant selectable marker for eukaryotic cells comprising the human
cytidine deaminase amino acid sequence as set forth in SEQ. ID NO: 2 or a
functional variant thereof, wherein said sequence confers resistance to
cytosine arabinoside or related cytosine analogues when expressed in said
eukaryotic cell.
Other features and advantages of the invention will be apparent from the
description of the preferred embodiments given hereinafter and from the
claims.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the construction of pMFG-CD;
FIG. 2 shows the polymerase chain reaction detecting the presence of the
cytidine deaminase cDNA in genomic DNA of GP+E86 cells transfected with
pMFG-CD;
FIG. 3 shows the plasmid DNA construct pGEX-CD containing the human
cytidine deaminase gene; and
FIG. 4 shows the expression of the CD protein using the expression vector
of FIG. 2.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates, in part, to the insertion of the human
cytidine deaminase cDNA into a eukaryotic expression vector. A principle
embodiment of this aspect of the present invention relates to the
successful expression of the CD cDNA in mammalian cells and the subsequent
resistance to the toxic effects of ARA-C and related cytosine nucleoside
analogs provided by the cDNA. The present invention also relates to
methods that apply to the above CD gene as a selectable marker in gene
transfer studies and therapies. In particular, the present invention
relates to the human cDNA for CD in an eukaryotic expression vector, for
example, pMFG and the expression of the cDNA in mammalian cells, for
example, murine fibroblasts.
In mammalian cells cytidine deaminase converts cytidine to uridine and
probably serves the purpose to maintain a balance of pyrimidine
nucleotides in the ribonucleotide and deoxyribonucleotide pools.
The distinguishing feature of the present invention is the expression of
the CD cDNA in eukaryotic cells for the purpose of creating a positive
selection system. One skilled in the art of molecular biology may express
the CD cDNA in a variety of eukaryotic expression vectors to achieve the
same purposes as those disclosed herein.
Introduction of the CD cDNA into mammalian cells results in the ability of
the mammalian transfected cells to convert cytidine to uridine; normally
most mammalian cells contain only low levels of the enzyme cytidine
deaminase. An increased in vitro conversion of radiolabelled cytidine to
uridine is consistently seen in transfected cells. The presence and
expression of the CD cDNA has no apparent deleterious effects upon the
cells. However, when such cells are exposed to ARA-C they show resistance
to inhibition of colony formation and to inhibition of DNA synthesis
produced by this cytosine nucleoside analog. Normal cells that do not
express or express low level of cytidine deaminase are sensitive to all
these inhibitory effects produced by ARA-C.
The present invention is the first to demonstrate that a eukaryotic enzyme
for cytidine deaminase (i.e.: the human enzyme) can, upon transfection
into cells, render them resistant to ARA-C. The transfection can be
performed by a great variety of methods including but not limited thereto
to calcium phosphate transfection, electroporation and retroviral
transfection. The nucleic acid sequences encoding CD can remain
unintegrated as part of an episome or can be inserted into the genome. By
providing a method of rendering eukaryotic cells, and more preferably
mammalian cells, resistant to ARA-C by virtue of the expression of the
cytidine deaminase cDNA, one skilled in the art will be enabled to apply
the cytidine deaminase positive selection system (CDPSS) to a variety of
tissues by simply applying known techniques in molecular biology and
retrovirology.
Another way to confer tissue specificity may be to deliver the CDPSS using
the pMFG-CD plasmid in different packaging cell lines, an example of such
a cell line is GP+E86. A variety of retrovirus packaging lines which have
different cell-type and species tropisms have been described (Cepko, 1989,
In Current Protocols in Molecular Biology, pp 9.10.1-9.11.12, Ausubel et
al., Eds., Wiley & Sons).
One skilled in the art of molecular biology will have no difficulty
adapting the teachings of this invention in order to transfect a different
packaging cell line, thereby potentially changing the target cell
specificity of the CDPSS.
The CDPSS system can be modified by someone skilled in the art to
incorporate the CDPSS and the therapeutic gene in the same vector. There
are several ways of accomplishing this modification. One example would be
to clone a therapeutic gene next to the CD cDNA in pMFG-CD. The expression
of both genes could be accomplished by the insertion of the internal
ribosomal entry site between the two genes (Ghattas et al., 1991, Mol.
Cell. Biol. 11: 5848-5859). Any cell altered by the vector would then
contain the CD cDNA and the therapeutic gene. Alternatively, the nucleic
acid sequence to be expressed can be harbored by a different vector than
that harboring the CD nucleic acid sequence, the two vectors can be
co-transfected using a variety of well known methods (In Current Protocols
in Molecular Biology, 1989, Ausubel et al., Eds., Wiley & Sons) The
co-transfected vector can be a viral or non-viral plasmid.
The present invention further relates to the creation of novel double
positive selection vectors. The CD nucleic acid sequence of the present
invention can be inserted along with the mutated human dihydrofolate
reductase gene (Corey et al., 1990, Blood 75: 337-343) into a gene
transfer vector. The cells receiving the vector are rendered resistant to
ARA-C or analogs thereof and methotrexate, thereby providing a double
positive selection system for protecting cells from the toxic effects of
two different antimetabolites. It is also contemplated herein, that a
double selection system can be dependent on the presence of the CD nucleic
acid sequence of the present invention and that of the mutated human
dihydrofolate reductase gene (DHFR), on different vectors. Similarly,
analogs of DHFR, or other dominant selectable markers can be used in a
double (or more) selection system. Other examples of dominant selectable
markers include but are not limited to a neomycin gene conferring
resistance to G418, the gpt gene, conferring resistance to mycophenolic
acid and a multidrug resistance gene, conferring resistance to a variety
of drugs such as adriamycin, colchicine and vincristine.
In another embodiment, the present invention relates to a therapeutic
method for the treatment of cancer, viral and immune diseases. This
invention relates to making normal bone marrow cells resistant to the
toxic effects of chemotherapy, for example ARA-C. This can be accomplished
by gene transfer of CD into normal bone marrow cells and transplantation
of these cells into the animal to be treated. It will then be possible to
administer higher doses of ARA-C to this animal to obtain a superior
therapeutic effect without encountering unacceptable bone marrow toxicity
(Corey et al., 1990 Blood, 75: 337-343; Podda et al., 1992, Proc. Natl.
Acad. Sci. USA 89: 9676-9680; and Ward et al., 1994, Blood. 84:
1408-1414). In a preferred embodiment, the animal is a human patient. In
another preferred embodiment, the human patient undergoes cancer therapy
with ARA-C or related cytosine analogs.
Thus, it is possible to make use of the discovery that expression of the
human cytidine deaminase cDNA is mammalian cells confers thereupon
resistance to a cytosine arabinoside or selected cytosine analogs in the
following four main contexts:
A. as a dominant selectable marker;
B. as a diagnostic tool;
C. in the design of new drugs; and
D. in gene therapy.
A. Dominant Selectable Marker
When properly expressed, the cDNA clone described allows the production of
a dominant selectable trait. Indeed, when the nucleic acid sequence as set
forth in SEQ. ID NO: 1, or a functional variant or homologue thereof, is
introduced into a drug-sensitive cell under conditions appropriate for its
expression, the drug-sensitive cell becomes drug resistant and is able to
survive and form colonies even in media containing drug levels which
normally cause death of drug-sensitive cells.
The cytidine deaminase nucleic acid sequence of the present invention can
also be used as a vehicle for moving hybrid genes into new host cells and
monitoring their presence. It is thus possible to engineer a DNA sequence
which includes the cytidine deaminase gene and at least one gene of
interest into a plasmid, and introduce this plasmid into a host cell,
monitoring the presence of the plasmid in these cells by culturing in
media containing cytosine arabinoside or related cytosine analogs. Cells
which contain the cytidine deaminase gene and the gene(s) of interest will
survive and those which do not will die.
B. Diagnostic Tool
The nucleic acid sequence of the cytidine deaminase or parts thereof can be
used to assess the level of expression of this gene in tissues or cells.
For example, the RNA can be isolated from tumour cells and through
Northern blot analysis (Sambrook et al., 1989) the level of expression of
the cytidine deaminase gene can be measured to assess the level of
expression thereof. Such an information can be advantageous for monitoring
the resistance of the tumour to the chemotherapeutic regimen. Another
method for measuring the level of expression includes in situ
hybridization.
Antibodies raised against cytidine deaminase protein can also be used in a
diagnostic context. Such antibodies, whether polyclonal or monoclonal can
be used in different types of immuno-assays to measure the level of
expression of cytidine deaminase protein.
Using large amounts of cytidine deaminase, obtained for example through the
expression of the pGEX-4T-1 vector, the protein or segment thereof can be
used in standard antibody production techniques using for example, a
rabbit or a rat as a host. If monoclonal antibodies are produced, the
well-known techniques involving fusion to form hybridomas would be used.
C. Design of New Drugs
The above information makes it possible to design new drugs or modify
presently available drugs to increase their activity toward drug-resistant
cells, or to design a cytidine deaminase which is more efficient at
detoxifying cytosine arabinoside or related cytosine analogs. In addition,
the present information makes it also possible to screen for inhibitors of
cytidine deaminase. For example, purification of large quantities of
mature cytidine deaminase will allow study of the three dimensional
structure thereof. Combined with the availability of multiple antibodies
directed towards segments of this protein, it will be possible to define
the specific mechanism imparting drug resistance and permit the design of
new drugs, or modification of old ones affecting the action of the
protein, or alternatively design modified protein interacting differently
with these drugs.
D. Gene Therapy
Because of the extensive depletion of bone marrow which occurs when a
patient is treated with chemotherapeutic drugs, the patient is unable to
fight-off a variety of bacterial, fungal and viral pathogens. The cDNA of
the present invention or variants thereof can be used to alleviate this
problem. This can be accomplished by inserting the cDNA described or
variants thereof into appropriate amphotropic or ecotropic retroviral
constructs which can then be used to infect normal bone marrow cells. This
approach can be valuable in a situation in which bone marrow is removed
from a patient, the gene inserted in vitro and the resulting engineered
marrow reintroduced into the patient (autologous treatment). Insertion of
heterologous marrow is also contemplated.
The treatment of the marrow can also be valuable in bone marrow
transplants, in which the patient's initial bone marrow is replaced by
marrow into which the cytidine deaminase cDNA or functional variant
thereof has been incorporated.
Thus, in these examples, the engineered bone marrow cells are resistant to
the effects of a chemotherapeutic regimen with cytosine arabinoside or
related cytosine analogs and are thus able to repopulate the depleted
marrow. The addition of further dominant selectable markers, adapted to a
particular chemotherapeutic regimen would also be beneficial. Thus, it
will be possible to administer higher concentrations of the
chemotherapeutic drug(s) to the individual without destroying the
transplanted bone marrow. This would contribute to the ability of the
patient to survive.
Molecular Biology Techniques
Plasmid pDR2 contains the complete cDNA sequence for human cytidine
deaminase (Laliberte et al., 1994, Cancer Res. 54: 5401-5407, the context
of which is incorporated by reference; Gene Bank accession number L27943).
Briefly, pDR2 was obtained by screening a human liver cDNA library in
lambdaDR2 phage (Clontech) with radiolabelled probes. The probes were
obtained by peptide sequencing from purified CD protein. Briefly, a
cytidine deaminase precipitate was obtained from homogenized human
placenta (fraction I). The precipitate was resuspended (fraction II),
incubated in a 75.degree. C. water bath and rapidly cooled. After
centrifugation, the supernatant was recovered and concentrated (Amicon
Centriprep-30.TM.) to give fraction III. Fractions III to VI were purified
by coloumn chromatography (Sephacryl S-200.TM. gel filtration,
PAER-1000.TM. ion exchange, and Mono-Q.TM. ion exchange chromatography at
pH 6.2 and 7.5). The final step used in the chromatography yielded a
single peak of CD activity to give fraction VII (Laliberte et al., 1994,
Cancer Res. 54: 5401-5407). Clostripain digestion or mild acid cleavage
permitted the generation of peptides, which were purified and submitted to
automated Edman degradation in a Porton Protein/Peptide
MicroSequencer.TM.. Some peptides were cleaved with cyanogen bromide prior
to sequencing. The amino acid sequences of one such peptide (E) permitted
the design of oligonucleotides.
PCR reaction using these different oligonucleotides were performed in the
presence of cDNA from the HL-60 human cDNA library. The PCR products were
purified using the Magic PCR Preps DNA Purification System.TM. (Promega)
and cloned in pCRII plasmid using the TA Cloning kit.TM. (Invitrogen).
Sequencing of a 470 bp PCR product harbored in pCRII permitted the
identification of a DNA sequence encoding the codons for peptide E
(Laliberte et al., 1994, Cancer Res. 54: 5401-5407). Sequencing was
carried out using the Sequenase.TM. 2.0 kit from USB. The sequencing data
obtained with this PCR product, permitted the design of 5' and 3'
oligonucleotide primers which were used to amplify by PCR a specific DNA
probe from liver cDNA (364 bp). This 364 bp probe was radiolabelled and
used to screen the HL-60 cDNA phage library mentioned above. One positive
clone was detected and converted to a plasmid (pDR2) using the method of
Clontech.
The insert size of this plasmid was estimated at approximately 950bp and
was sequenced in both orientation by the chain termination method using
fluoro-dATP and a Pharmacia A.L.F. automatic sequencer.TM.. The 910-bp
clone contained 117-bp 5'-nontranslated sequence, an open reading frame of
438-bp, and a 336-bp 3'-nontranslated region ending with a poly(A).sup.+
tail (SEQ. ID NO: 1). The translated region predicts an open reading frame
of 146 codons (SEQ. ID NO: 2) having a [predicted molecular mass of 16.2
kDa. For the plasmid pMFG-tPA the Moloney murine leukemia virus long
terminal repeat (LTR) sequences are used to generate both a full length
viral RNA (for encapsidation into virus particles) and a subgenomic mRNA
which is responsible for the expression of inserted sequences (Dwarki et
al., 1995, Proc. Natl. Acad. Sci. USA, 92: 1023-1027; and Guild et al.,
WO92/07943, published May 14, 1992). Protein coding sequences are inserted
between the NcoI and BamHI sites. No selectable marker exists in the
vector. The pSV-neo vector contains the SV40 early promoter and the
neomycin phosphotransferase (neo) gene. The neo gene allows cells to
survive in the presence of the protein synthesis inhibitor, neo or its
analog G418 (In Current Protocols in Molecular Biology, 1989, Ausubel et
al. Eds., John Wiley & Sons). These vectors and the subsequent constructs
are depicted in FIG. 1.
Oligonucleotides were synthesized on a Pharmacia Gene Assembler Plus.TM.
Instrument (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual,
2nd, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y).
Oligonucleotides 5'-TAC CAC CAT GGC CCA GAA GCG T-3' (SEQ. ID NO: 3) and
5'-TGG GCA GGA TCC GGC TGT CAC T-3' (SEQ. ID NO: 4) were used as primers
and pDR-2 as the DNA template in a polymerase chain reaction (PCR) using
the MJ Research thermocycler. SEQ. ID NO: 3 contains a NcoI recognition
site at its 5' end as well as the first 5 codons of the human CD cDNA in
the sense orientation. SEQ. ID NO: 4 contains a BamHI recognition site at
its 5' end and the last 2 codons of the human CD cDNA in the antisense
orientation. The 465 bp amplified DNA and pMFG-tPA were digested with NcoI
and BamHI, purified by agarose gel electrophoresis and ligated with T4 DNA
ligase according to known methods (Sambrook et al., 1989). Competent E.
coli were transformed with the construct and individual colonies of
transformants were screened for insertion by PCR and restriction enzyme
digests, following standard procedures. Large scale preparation of
plasmids were produced by standard methods and the plasmids purified by
Quiagen.TM. columns (J. Sambrook et al., 1989). The resulting plasmid with
the CD cDNA is called pMFG-CD. The 5' region of the cDNA was sequenced by
the dideoxynucleotide chain termination method using a Pharmacia Automatic
DNA Sequencer.TM. with fluoro-dATP to verify the sequence (In Current
Protocols in Molecular Biology, 1989, Ausubel et al., Eds. Wiley & Sons).
In order to verify the integration of the CD cDNA into the genomic DNA of
the transfected cells the oligonucleotide 5'-GGT GGA CCA TCC TCT AGA
CTG-3' (SEQ. ID NO: 5) and 5'-AGC AGC TCC TGG ACC GTC ATG-3' (SEQ. ID NO:
6) were used as primers with .about.1 ng of genomic DNA in the PCR to
amplify a specific 421 bp fragment, as predicted by the DNA sequence of
the pMFG-CD construct. The sense oligonucleotide SEQ. ID NO: 5 was
.about.70 bp downstream from the splice acceptor (SA) region of MFG. The
antisense oligonucleotide SEQ. ID NO: 6 was from positions 377-397 of the
CD coding region. Genomic DNA was isolated from the GP+E86 cells with In
ViSorb DNA Kit.TM. (ID Laboratory) by cell lysis with guanidine
thiocyanate and DNA adsorption on silica gel (In Current Protocols in
Molecular Biology, 1989).
In order to clone the CD cDNA in a bacterial expression vector the coding
region from pDR-2 was amplified by PCR using oligonucleotides 5'-ACG GGA
TCC ATG GCC CAG AAG CGT CCT G-3' (SEQ. ID NO: 7) and 5'-CCG CTC GAG TCA
CTG AGT CTT CTG CAG-3' (SEQ. ID NO: 8). SEQ. ID NO: 7 contains a BamHI
site at the 5' end and SEQ. ID NO: 8 contains a XhoI site at its 5' end.
The amplified DNA was digested with BamHI and XhoI and cloned into
pGEX-4T-1 to give pGEX-CD. This DNA construct was used to transform E.
coli and the fusion protein glutathione-S-transferase-cytidine deaminase
was induced with isopropyl-.beta.-D-thiogalatoside and purified on a
glutathione affinity chromatographic column. Glutathione-S-transferase was
removed from the fusion protein by cleavage with thrombin to give the
purified cytidine deaminase (Laliberte et al., 1994, Cancer Res. 54:
5401-5407).
Cell Culture Techniques
Cells were grown in DMEM medium supplemented with 10% heat-inactivated
fetal calf serum and 5 .mu.g/ml Gentamycin (DM-10S medium) and incubated
at 37.degree. C. and 5% CO.sub.2. NIH 3T3 cells, a murine fibroblast cell
line which is used frequently in retroviral transduction experiments (In
Current Protocols in Molecular Biology, 1989). GP+E86, murine ecotropic
packaging cells were described previously (Markowitz et al., 1988, J.
Virol. 62: 1120-1124). GP+86E cells were derived from NIH-3T3 cells and
contain a stably integrated incompetent retroviral genome; it functions as
a retroviral packaging cell line when transfected with plasmids containing
a sequence encoding a retroviral mRNA with an intact packaging signal.
Plasmid pMFG-CD contains retroviral LTR and an intact packaging signal.
GP+E86 cells were transfected with the purified plasmid DNAs pMFG-CD and
pSV-neo using the standard calcium phosphate precipitation method (In
Current Protocols in Molecular Biology, 1989). 72 hours post-transfection,
G418 at 400 .mu.g/ml was added to the medium and the cells were selected
in this medium for 14 days. Clones of cells resistant to G418 were
isolated by ring cloning or by dilution (Cepko, 1989). Thereafter the
cells were maintained in D-10S medium only.
For viral transduction the supernatant from clones of GP+E86 cells was
added to the 3T3 cells (Cepko, 1989). 72 hours post-transduction, cytosine
arabinoside (ARA-C) at 5 .mu.M was added to the medium and the cells
selected in this medium for 14-21 days. Clones resistant to ARA-C were
isolated as described above.
Clonogenic assays were performed as follows. The cells were diluted to 100
cells/ml and 1 ml placed in wells of 12-well Costar.TM. dish. 18-20 hours
later, ARA-C was added at the concentrations described in the examples and
tables below, and the incubation continued for an additional 15 days. The
wells were then stained with 0.5% methylene blue in 50% methanol, and
colonies of greater than 10.sup.3 cells were counted. DNA synthesis assays
were performed as follows. Cells were diluted to 10.sup.4 cells/ml and 1
ml placed in a 12-well Costar dish. After incubation for 4 days, ARA-C was
added at the concentrations indicated in the examples and tables below.
After an incubation for an additional 16 hours, 0.5 .mu.Ci of .sup.3
H-thymidine (20 Ci/mmol) was added and incubated for a further 4 hours.
The amount of radioactivity incorporated into DNA was determined by known
methods.
Enzyme Assay
In vitro assay for cytosine deaminase was performed using a modification of
a previously described method (Laliberte et al., 1994, Cancer Res. 54:
5401-5407). Briefly, 5.times.10.sup.7 monolayer cells were trypsinized,
centrifuged and washed once in phosphate buffered saline (PBS),
recentrifuged and suspended in 100 .mu.l of 20 mM TrisCl pH 8.0 and 5 mM
dithiothreitol. The cell suspension was then subjected to 3 cycles of
rapid freezing and thawing. The mixture was centrifuged at maximum speed
in a tabletop microfuge at 5.degree. C. for 5 min. The supernatant was
used in an assay to measure the conversion of .sup.3 H-cytidine to .sup.3
H-uridine.
The present invention will be more readily understood by referring to the
following examples which are given to illustrate the invention rather than
to limit its scope.
EXAMPLE 1
Cloning of Human Cytidine Deaminase cDNA
FIG. 1 summarizes the cloning process. Cloning of the human cytosine
deaminase gene into the eukaryotic expression vector pMFG was performed in
the following way. The plasmid pDR-2 containing the cDNA for CD was used
as the template for the polymerase chain reaction (PCR). Using a sense
primer containing NcoI linker and an antisense primer containing BamHI
linker the 438 bp coding region of CD was amplified. The amplified DNA was
digested with NcoI and BamHI, purified and cloned into pMFG. The resulting
construct was named pMFG-CD. Sequencing of the 5' region of the cytidine
deaminase cDNA confirmed the presence of the desired sequence and the
start site in pMFG-CD. The sequence of pMFG-CD in the 5'-region of CD is
shown. FIG. 1 summarizes the important features from pMFG-CD and contains
among other eukaryotic expression elements, the LTR promoter promoting the
cytidine deaminase cDNA, and a packaging sequence (necessary for
encapsidation of the viral RNA into the virus particle).
EXAMPLE 2
Transfection of Mammalian Cells With pMFG-CD Results in Expression of the
Cytidine Deaminase cDNA
GP+E86 cells were transfected with pMFG-CD and pSV-neo and 72 hours later
placed in medium containing G418 0.5 mg/ml. The cells were incubated in
G418 for 14-21 days and then maintained in regular medium. Resistance to
the neomycin analog G418 allowed for the enrichment of the population of
cells that had taken up the plasmid sequences. These cells were then
exposed to ARA-C 5 .mu.M for 14 days and the surviving cells that showed
resistance to this cytosine nucleoside analog were cloned to give lines
GP+86E-CD3 and GP+86E-CD4.
Incorporation of the cytidine deaminase cDNA into the genome was
demonstrated by genetic analysis of the DNA and the expression of the
enzyme. PCR reactions employing primers corresponding to the gag region of
pMFG and the coding region of CD were used to amplify the specific DNA of
421 bp as predicted by DNA sequence analysis. FIG. 2 shows the PCR product
produced using cellular DNA as template for MFG-CD DNA synthesis. Briefly,
genomic DNA (.about.1 ng) underwent PCR using a sense primer from the
5'-LTR region of MFG and an antisense primer from the coding region of CD.
As predicted by the sequence analysis, the amplified DNA had a size of 421
bp. Genomic DNA from non-transfected cells failed to show bands of
amplified DNA with the same primers.
These cell populations were also assayed for the expression of cytidine
deaminase cDNA. An in vitro assay measured the conversion of radiolabelled
cytosine to uridine in lysates of cells. GP+86E-CD3 and GP+86E-CD4 cells
showed high levels of cytidine deaminase activity while the
non-transfected control cell lines did not (Table 1).
TABLE 1
______________________________________
Conversion of cytidine to uridine in vitro by lysates
of cell lines containing the cytidine deaminase gene
Cytidine deaminase activity (units/mg).sup.(1)
Cell line Exp. 1 Exp. 2
______________________________________
GP + E-86 1.0
GP + E-86-lac 4 0.7
GP + E-86-CD3 182.8
GP + E-86-CD4 100.7
______________________________________
.sup.(1) Units of activity is defined as nmoles deaminated per min.
EXAMPLE 3
Cell Lines Expressing the Cytidine Deaminase CDNA are Resistant to ARA-C
Inhibitory Effects and Toxicity
Clonogenic assays were performed to assess the sensitivity of cells to
ARA-C. 100 cells were inoculated into tissue culture dish wells and after
a 12 day exposure to different concentrations of ARA-C, the number of
colonies were counted. Drug resistant cells that can survive and
proliferate under these conditions can give rise to individual colonies.
The inoculum was dilute enough to allow easy identification and
enumeration of individual colonies.
Table 2 demonstrates that cells expressing the cytidine deaminase cDNA can
give rise to colonies in the presence of toxic concentrations of ARA-C,
due to the inactivation of this cytosine nucleoside analog by deamination.
In contradistinction thereto however, cells which do not express the
cytidine deaminase cDNA do not form colonies in the presence of toxic
concentrations of ARA-C. Indeed, at concentrations of ARA-C of 10.sup.-6 M
and higher, cell lines GP+E86-CD3, GP+E86-CD4 and 3T3-CD3-V5 were able to
form a significant number of colonies indicating drug resistance to ARA-C,
while under these same conditions, the control cell lines GP+E86 and 3T3
did not form any colonies.
TABLE 2
______________________________________
Inhibition of colony
formation by different conc.
of ARA-C
Average colony count.sup.(1)
______________________________________
Conc. of ARA-C (.mu.M)
Cell line Exp. No. 0 0.1 1.0 10.0
______________________________________
GP + 86 #1 28 33 0 0
#2 33 29 0 0
GP + 86-CD3 #1 18 25 19 19
#2
GP + 86-CD4 #1 25 23 24 3
#2
3T3 #1 27 0 0 0
#2 31 27 0 0
3T3-CD3-V5 #1 31 34 28 7
#2
______________________________________
.sup.(1) Cells were exposed to the indicated concentrations of ARAC for 1
days
Since ARA-C is a potent inhibitor of DNA synthesis, it was important to
assess the inhibitory activity of this analog on DNA synthesis in cells
expressing CD versus cells not expressing it.
DNA synthesis was measured by the incorporation of radiolabelled thymidine
into cellular DNA. As shown in Table 3, ARA-C does not have a very
effective inhibitory activity on DNA synthesis in cells expressing the
cytidine deaminase cDNA. In cells not expressing the cytidine deaminase
cDNA, however, a significant inhibition of DNA synthesis is observed in
the presence of ARA-C. Indeed, Table 3 shows that ARA-C at concentration
of 10.sup.-6 M and greater, only produced a very weak inhibition of DNA
synthesis on cell lines GP+E86-CD3, GP+E86-CD4 and 3T3-CD3-V5. In
contrast, in the control cell lines GP+E86 and 3T3, ARA-C produced a
potent inhibition of DNA synthesis.
TABLE 3
______________________________________
Inhibition 3H-thymidine incorporation by ARA-C in
cell lines expressing cytidine deaminase gene
Cell line
Additive cpm .+-. sem.sup.(1)
Inhibition (%)
______________________________________
GP + 86 none 1,930 --
ARA-C 0.5 .mu.m 2,089 <1
ARA-C 1.0 .mu.m 1,141 40.9
ARA-C 5.0 .mu.m 551 71.5
ARA-C 10 .mu.m 316 83.6
GP + 86-CD3 none 6,374 --
ARA-C 0.5 .mu.m 5,831 8.5
ARA-C 1.0 .mu.m 6,030 5.4
ARA-C 5.0 .mu.m 5,348 16.1
ARA-C 10 .mu.m 5,149 19.2
GP + 86-CD4 none 2,620 --
ARA-C 1.0 .mu.m 2,911 <1
ARA-C 10 .mu.m 3,321 <1
______________________________________
.sup.(1) cells were incubated with ARAC for 16 hr followed by addition of
.sup.3 Hthymidine for an additional 5 hr. The numbers indicated have been
averaged.
EXAMPLE 4
Retroviral Mediated Gene Transfer Results in Successful Expression of the
Cytidine Deaminase cDNA
3T3 cells were transduced by exposure to retroviral particles present in
the supernatant from GP+E86-CD3 packaging cells and selected in medium
containing ARA-C 5 .mu.M as described in material and methods. The
resulting cell line was designated 3T3-CD3-V5. As seen in Table 1, lysates
from 3T3-CD3-V5 contained very high levels of cytidine deaminase activity
as compared to the control 3T3 cells. The 3T3-CD3-V5 cells were also
resistant to ARA-C in clonogenic assays (Table 2), and in DNA synthesis
assays (Table 3).
EXAMPLE 5
Expression of Cytidine Deaminase in Prokaryotes
FIG. 3 shows the plasmid pGEX-CD bacterial expression vector. E. coli was
transformed with pGEX-CD, the fusion protein
glutathione-S-transferase-cytidine deaminase was induced with
isopropyl-.beta.-D-thiogalatoside (IPTG) and purified on a glutathione
affinity chromatographic column. FIG. 4 shows a sodium dodecyl
sulfate-polyacrylamide gel electrophoresis profile of the expression of CD
in E. coli. Lane 1 shows the induction of glutathione-S-transferase (GST)
(27.5 kDa) induced with IPTG in E. coli transformed with pGEX-4T, a vector
which does not contain CD. Lane 2 shows the induction by IPTG of the
fusion protein glutathione-S-transferase-cytidine deaminase (GST-CD) (42.4
kDa) from E. coli transformed with pGEX-CD. Lane 3 shows the purified
cytidine deaminase (CD) (16.3 kDa) after cleavage of the fusion protein
shown in lane 2 with thrombin.
While the invention has been described with particular reference to the
illustrated embodiment, it will be understood that numerous modifications
thereto will appear to those skilled in the art. Accordingly, the above
description and accompanying drawings should be taken as illustrative of
the invention and not in a limiting sense.
__________________________________________________________________________
# SEQUENCE LISTING
- - - - (1) GENERAL INFORMATION:
- - (iii) NUMBER OF SEQUENCES: 8
- - - - (2) INFORMATION FOR SEQ ID NO:1:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 892 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (iii) HYPOTHETICAL: NO
- - (iv) ANTI-SENSE: NO
- - (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapi - #ens
(F) TISSUE TYPE: Myeloi - #d
(H) CELL LINE: HL-60
- - (viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: 1
- - (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 118..558
- - (x) PUBLICATION INFORMATION:
(A) AUTHORS: Laliberte, - #Josee
Momparler, - #Richard L
(B) TITLE: Human Cytidi - #ne Deaminase: Purification of
Enzyme, C - #loning, and Expression of its
complementar - #y DNA
(C) JOURNAL: Cancer Res - #earch
(D) VOLUME: 54
(F) PAGES: 5401-5407
(G) DATE: October 15-19 - #94
(K) RELEVANT RESIDUES I - #N SEQ ID NO:1: FROM 1 TO 922
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
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#GGGCCCCA 60
- - GCCAGGCTGG CCGGAGCTCC TGTTTCCGCT GCTCTGCTGC CTGCCCGGGG TA - #CCAAC
117
- - ATG GCC CAG AAG CGT CCT GCC TGC ACC CTG AA - #G CCT GAG TGT GTC CAG
165
Met Ala Gln Lys Arg Pro Ala Cys Thr Leu Ly - #s Pro Glu Cys Val Gln
1 5 - # 10 - # 15
- - CAG CTG CTG GTT TGC TCC CAG GAG GCC AAG AA - #G TCA GCC TAC TGC CCC
213
Gln Leu Leu Val Cys Ser Gln Glu Ala Lys Ly - #s Ser Ala Tyr Cys Pro
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- - TAC AGT CAC TTT CCT GTG GGG GCT GCC CTG CT - #C ACC CAG GAG GGG AGA
261
Tyr Ser His Phe Pro Val Gly Ala Ala Leu Le - #u Thr Gln Glu Gly Arg
35 - # 40 - # 45
- - ATC TTC AAA GGG TGC AAC ATA GAA AAT GCC TG - #C TAC CCG CTG GGC ATC
309
Ile Phe Lys Gly Cys Asn Ile Glu Asn Ala Cy - #s Tyr Pro Leu Gly Ile
50 - # 55 - # 60
- - TGT GCT GAA CGG ACC GCT ATC CAG AAG GCC GT - #C TCA GAA GGG TAC AAG
357
Cys Ala Glu Arg Thr Ala Ile Gln Lys Ala Va - #l Ser Glu Gly Tyr Lys
65 - # 70 - # 75 - # 80
- - GAT TTC AGG GCA ATT GCT ATC GCC AGT GAC AT - #G CAA GAT GAT TTT ATC
405
Asp Phe Arg Ala Ile Ala Ile Ala Ser Asp Me - #t Gln Asp Asp Phe Ile
85 - # 90 - # 95
- - TCT CCA TGT GGG GCC TGC AGG CAA GTC ATG AG - #A GAG TTT GGC ACC AAC
453
Ser Pro Cys Gly Ala Cys Arg Gln Val Met Ar - #g Glu Phe Gly Thr Asn
100 - # 105 - # 110
- - TGG CCC GTG TAC ATG ACC AAG CCG GAT GGT AC - #G TAT ATT GTC ATG ACG
501
Trp Pro Val Tyr Met Thr Lys Pro Asp Gly Th - #r Tyr Ile Val Met Thr
115 - # 120 - # 125
- - GTC CAG GAG CTG CTG CCC TCC TCC TTT GGG CC - #T GAG GAC CTG CAG AAG
549
Val Gln Glu Leu Leu Pro Ser Ser Phe Gly Pr - #o Glu Asp Leu Gln Lys
130 - # 135 - # 140
- - ACT CAG TGA CAGCCAGAGA ATGCCCACTG CCTGTAACAG CCACCTGGA - #G
598
Thr Gln *
145
- - AACTTCATAA AGATGTCTCA CAGCCCTGGG GACACCTGCC CAGTGGCCCC AG -
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- - GACTGGGCAA AGATGATGTT TCCAGATTAC ACTCCAGCCT GAGTCAGCAC CC -
#CTCCTAGC 718
- - AACCTGCCTT GGGACTTAGA ACACCGCCGC CCCCCTGCCC CACCTTTCCT TT -
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- - TGGGCCCTCT TTCAAAGTCC AGCCTAGTCT GGACTGCTTC CCCATCAGCC TT -
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- - TCTATCCTGT TCCGAGCAAC TTTTCTAATT ATAAACATCA CAGAACATCC TG - #GA
892
- - - - (2) INFORMATION FOR SEQ ID NO:2:
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(B) TYPE: amino acid
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
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1 5 - # 10 - # 15
- - Gln Leu Leu Val Cys Ser Gln Glu Ala Lys Ly - #s Ser Ala Tyr Cys Pro
20 - # 25 - # 30
- - Tyr Ser His Phe Pro Val Gly Ala Ala Leu Le - #u Thr Gln Glu Gly Arg
35 - # 40 - # 45
- - Ile Phe Lys Gly Cys Asn Ile Glu Asn Ala Cy - #s Tyr Pro Leu Gly Ile
50 - # 55 - # 60
- - Cys Ala Glu Arg Thr Ala Ile Gln Lys Ala Va - #l Ser Glu Gly Tyr Lys
65 - # 70 - # 75 - # 80
- - Asp Phe Arg Ala Ile Ala Ile Ala Ser Asp Me - #t Gln Asp Asp Phe Ile
85 - # 90 - # 95
- - Ser Pro Cys Gly Ala Cys Arg Gln Val Met Ar - #g Glu Phe Gly Thr Asn
100 - # 105 - # 110
- - Trp Pro Val Tyr Met Thr Lys Pro Asp Gly Th - #r Tyr Ile Val Met Thr
115 - # 120 - # 125
- - Val Gln Glu Leu Leu Pro Ser Ser Phe Gly Pr - #o Glu Asp Leu Gln Lys
130 - # 135 - # 140
- - Thr Gln
145
- - - - (2) INFORMATION FOR SEQ ID NO:3:
- - (i) SEQUENCE CHARACTERISTICS:
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(B) TYPE: nucleic acid
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(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc - #= "dna"
- - (iii) HYPOTHETICAL: NO
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
- - TACCACCATG GCCCAGAAGC GT - # - #
22
- - - - (2) INFORMATION FOR SEQ ID NO:4:
- - (i) SEQUENCE CHARACTERISTICS:
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- - (iii) HYPOTHETICAL: NO
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
- - TGGGCAGGAT CCGGCTGTCA CT - # - #
22
- - - - (2) INFORMATION FOR SEQ ID NO:5:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc - #= "dna"
- - (iii) HYPOTHETICAL: NO
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
- - GGTGGACCAT CCTCTAGACT G - # - #
- #21
- - - - (2) INFORMATION FOR SEQ ID NO:6:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc - #= "dna"
- - (iii) HYPOTHETICAL: NO
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
- - AGCAGCTCCT GGACCGTCAT G - # - #
- #21
- - - - (2) INFORMATION FOR SEQ ID NO:7:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc - #= "dna"
- - (iii) HYPOTHETICAL: NO
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
- - ACGGGATCCA TGGCCCAGAA GCGTCCTG - # - #
28
- - - - (2) INFORMATION FOR SEQ ID NO:8:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc - #= "dna"
- - (iii) HYPOTHETICAL: NO
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
- - CCGCTCGAGT CACTGAGTCT TCTGCAG - # - #
27
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